1. Field of the Invention
This invention relates to novel multibinding compounds (agents) that are macrolide antibiotics, aminoglycosides, lincosamides, oxazolidinones, streptogramins, tetracyclines or other compounds which bind to bacterial ribosomal RNA or one or more proteins involved in ribosomal protein synthesis in the bacterium, and to pharmaceutical compositions comprising such compounds. The compounds are useful as antibacterial agents for treating a variety of bacterial infections.
2. State of the Art
Organisms generate polypeptides (proteins) in order to survive. Organisms that cannot generate proteins cannot maintain viability. Because the majority of genes encode proteins, xe2x80x9cgene expressionxe2x80x9d is nearly synonymous with protein synthesis. Gene expression involves two stepsxe2x80x94transcription and translation. Genes code for proteins using various codons (units of three nucleotides), such as start codons (which initiate translation), stop codons (which stop translation) and codons in between the start and stop codons which selectively code for the various amino acids.
Translation is the RNA directed synthesis of polypeptides. This process requires all three classes of RNA. The template for correct addition of individual amino acids is the mRNA, yet both tRNAs and rRNAs are involved in the process.
Prokaryotes and eukaryotes use ribosomes to generate proteins. Ribosomes are cytoplasmic organelles, and are large complexes of proteins and three (prokaryotes) or four (eukaryotes) rRNA (ribosonial ribonucleic acid) molecules called subunits made in the nucleolus. Ribosomes serve as the site of mRNA translation. Once the two (large and small) subunits are joined by the mRNA from the nucleus, the ribosome translates the mRNA into a specific sequence of amino acids, or a polypeptide chain.
In its inactive state, the ribosome exists as two subunits; a large subunit and a small subunit. When the small subunit encounters an mRNA, the process of translation of the mRNA to protein begins. There are two sites in the large subunit, for subsequent amino acid-charged tRNAs to bind to and thus be close enough to each other for the formation of a peptide bond. The A site accepts a new tRNA bearing an amino acid, and the P site bears the tRNA attached to the growing chain.
tRNA (transfer RNA) is a specific RNA molecule which acts as a translator between mRNA and protein. Each tRNA has a specific anticodon and acceptor site. Each tRNA also has a specific charger protein; this protein can only bind to that particular tRNA and attach the correct amino acid to the acceptor site. The energy to make this bond comes from ATP. These charger proteins are called aminoacyl tRNA synthetases. The tRNAs carry activated amino acids into the ribosome. The ribosome is associated with the mRNA ensuring correct access of activated tRNAs and containing the necessary enzymatic activities to catalyze peptide bond formation.
Protein synthesis proceeds from the N-terminus to the C-terminus of the protein. The ribosomes xe2x80x9creadxe2x80x9d the mRNA in the 5xe2x80x2 to 3xe2x80x2 direction. Active translation occurs on polyribosomes (also termed polysomes). This means that more than one ribosome can be bound to and translate a given mRNA at any one time. Chain elongation occurs by sequential addition of amino acids to the C-terminal end of the ribosome bound polypeptide. Translation proceeds in an ordered process. First accurate and efficient initiation occurs, then chain elongation and finally accurate and efficient termination must occur. All three of these processes require specific proteins, some of which are ribosome associated and some of which are separate from the ribosome, but may be temporarily associated with it.
Initiation of Translation
Before translation occurs, a ribosome must dissociate into its 30S and 50S subunits. A ternary complex termed the preinitiation complex is formed consisting of the initiator, GTP, eIF-2 and the 40S subunit. The mRNA is bound to the preinitiation complex.
Initiation of translation in both prokaryotes and eukaryotes requires a specific initiator tRNA (which includes methionine). The initiation of translation requires recognition of a start (AUG) codon.
Peptide bond formation is catalyzed by a 23S rRNA component of the 50S subunit (peptidyl transferase). Another important site for protein synthesis is the 16S rRNA component of the 30S subunit (the aminoacyl tRNA site). When protein synthesis is terminated, release factor proteins bind to the stop codons, GTP hydrolysis occurs, and peptidyl transferase activity is stimulated, causing release of the protein from the tRNA.
Elongation of the New Protein
After the first charged tRNA appears in the A site, the ribosome shifts so that the tRNA is in the P site. New charged tRNAs, corresponding to the codons of the mRNA, enter the A site, and a peptide bond is formed between the two amino acids. The first tRNA is now released and the ribosome shifts again so that a tRNA carrying two amino acids is now in the P site, and a new charged tRNA can bind to the A site. This process of elongation continues until the ribosome reaches a stop codon.
Elongation requires specific non-ribosomal proteins. Elongation of polypeptides occurs in a cyclic manner. At the end of one complete round of amino acid addition the A site will be empty and ready to accept the incoming aminoacyl-tRNA dictated by the next codon of the mRNA. This means that not only does the incoming amino acid need to be attached to the peptide chain but the ribosome must move down the mRNA to the next codon. Each incoming aminoacyl-tRNA is brought to the ribosome by an eEF-1a-GTP complex. When the correct tRNA is deposited into the A site the GTP is hydrolyzed and the eEF-1a-GDP complex dissociates. For additional translocation events to occur the GDP must be exchanged for GTP. This is carried out by eEF-1bg similarly to the GTP exchange that occurs with eIF-2 catalyzed by eIF-2B. The peptide attached to the tRNA in the P site is transferred to the amino group at the aminoacyl-tRNA in the A site. This reaction is catalyzed by peptidyltransferase in a process termed transpeptidation. The elongated peptide now resides on a tRNA in the A site. The A site needs to be freed in order to accept the next aminoacyl-tRNA. The process of moving the peptidyl-tRNA from the A site to the P site is termed, translocation. Translocation is catalyzed by eEF-2 coupled to GTP hydrolysis. In translocation, the ribosome is moved along the mRNA such that the next codon of the mRNA resides under the A site. Following translocation, eEF-2 is released from the ribosome and the cycle can start over again.
Termination of the Protein
When the ribosome reaches a stop codon, no aminoacyl tRNA binds to the empty A site. This signals the ribosomes to break into its large and small subunits, releasing the new protein and the mRNA. The protein may then undergo post-translational modifications. For example, it might be cleaved by a proteolytic (protein-cutting) enzyme at a specific place, have some of its amino acids altered, or become phosphorylated or glycosylated.
Protein Synthesis Inhibitors
In bacteria, if the ribosomal RNA is inactivated, for example, through binding of a ligand to the ribosomal RNA, protein synthesis is adversely affected and the bacteria will likely die. Many of the antibiotics, in particular, MLS antibiotics, used to treat bacterial infections function by inhibiting translation. Inhibition can be effected at all stages of translation, from initiation to elongation to termination. Many antibiotics are believed to primarily attach to the ribosomal RNA at the 16S and 23S ribosomal subunits and inhibit growth of bacteria by inhibiting protein synthesis.
Chloramphenicol inhibits prokaryotic peptidyl transferase. Streptomycin and neomycin inhibit prokaryotic peptide chain initiation, also induce mRNA misreading. Tetracycline inhibits prokaryotic aminoacyl-tRNA binding to the ribosome small subunit. Erythromycin inhibits prokaryotic translocation through the ribosome large subunit, and fusidic acid functions in a manner similar to erythromycin.
MLS antibiotics such as erythromycin have been used for years to treat various infections, for example, those caused by gram positive bacteria, gram negative bacteria and anaerobic bacteria. Many bacteria are becoming drug resistant. One theory of how the bacteria become drug resistant is that their ribosomal RNA mutates such that the antibiotics no longer bind as ligands to the RNA.
A number of macrolide antibiotics are orally bioavailable, but in contact with the gastrointestinal tract, degrade to some degree to form products which cause adverse side effects such as diarrhea. For this reason, many derivatives of naturally occurring macrolide antibiotics such as erythromycin are esterified at the 6-position to minimize formation of the degradation products following oral administration.
It would be advantageous to provide agents useful as antibiotics which have higher bioavailability, and are less subject to degradation and which have improved affinity for ribosomal RNA. The present invention provides such agents.
This invention is directed to novel multibinding compounds (agents) that are macrolide antibiotics, aminoglycosides, lincosamides, oxazolidinones, streptogramins, tetracyclines or other compounds which bind to ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium. The multibinding compounds of this invention are useful as antibacterials, in particular, for gram positive, gram negative and anaerobic bacteria.
Accordingly, in one of its composition aspects, this invention provides a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a macrolide antibiotic, aminoglycoside, lincosamide, oxazolidinone, streptogramin, tetracycline or other compound which binds to ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium and adversely affects protein synthesis, and pharmaceutically-acceptable salts thereof.
In another of its composition aspects, this invention provides a multibinding compound of formula I:
(L)p(X)qxe2x80x83xe2x80x83I
wherein each L is independently a ligand comprising a macrolide antibiotic, aminoglycoside, lincosamide, oxazolidinone, streptogramin, tetracycline or other compound which binds to ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium; each X is independently a linker; p is an integer of from 2 to 10; and q is an integer of from 1 to 20; and pharmaceutically-acceptable salts thereof.
Preferably, q is less than p in the multibinding compounds of this invention.
Preferably, each ligand, L, in the multibinding compound of formula I is independently selected from the group consisting of erythromycin and ester prodrugs/derivatives thereof, such as erythromycin stearate and erythromycin estolate; clarithromycin, roxythromycin, azithromycin, aureomycin, oleandomycin, sulfisoxazole, spiramycin, troleandomycin, josamycin, cytovaricin, linezolid, eperezolid, clindamycin [AntirobeR], lincomycin, quinupristin, dalfopristin (Synercid, Rhone-Poulenc Rorer), streptomycin, amikacin, gentamicin, kanamycin, neomycin, tobramycin, netilmicin, paromomycin, tetracycline, chlortetracycline, doxycycline, minocycline, declomycin, methacycline, spectinomycin, and oxytetracycline, and analogues thereof, which are well known to those of skill in the art. Examples of suitable analogues include alkylated, esterified, amidated, alkoxylated, sulfonated, carboxylated, halogenated, phosphorylated, thiolated and hydroxylated analogues.
In still another of its composition aspects, this invention provides a multibinding compound of formula II:
Lxe2x80x2xe2x80x94Xxe2x80x2xe2x80x94Lxe2x80x2xe2x80x83xe2x80x83II
wherein each Lxe2x80x2 is independently a ligand comprising a macrolide antibiotic, aminoglycoside, lincosamide, oxazolidinone, streptogramin, tetracycline or other compound which binds to ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium and Xxe2x80x2 is a linker; and pharmaceutically-acceptable salts thereof.
Preferably, in the multibinding compound of formula II, each ligand, Lxe2x80x2, is independently selected from the group consisting of macrolide antibiotics, oxazoldinones, lincosamides, streptogranims, tetracyclines and aminoglycosides. and Xxe2x80x2 is a linker; and pharmaceutically-acceptable salts thereof.
Preferably, in the above embodiments, each linker (i.e. , X, Xxe2x80x2 or Xxe2x80x3) independently has the formula:
xe2x80x94Xaxe2x80x94Zxe2x80x94(Yaxe2x80x94Z)mxe2x80x94Ybxe2x80x94Zxe2x80x94Xaxe2x80x94
wherein
m is an integer of from 0 to 20;
Xa at each separate occurrence is selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)NRxe2x80x94, xe2x80x94C(S), xe2x80x94C(S)Oxe2x80x94, xe2x80x94C(S)NRxe2x80x94 or a covalent bond where R is as defined below;
Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;
Ya and Yb at each separate occurrence are selected from the group consisting of xe2x80x94C(O)NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2C(O)xe2x80x94, xe2x80x94NRxe2x80x2C(O)NRxe2x80x2xe2x80x94, xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(xe2x95x90NRxe2x80x2)xe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Nxe2x95x90C(Xa)xe2x80x94NRxe2x80x2-, xe2x80x94P(O)(ORxe2x80x2)xe2x80x94Oxe2x80x94, xe2x80x94S(O)nCRxe2x80x2Rxe2x80x3xe2x80x94, xe2x80x94S(O)nxe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94Sxe2x80x94Sxe2x80x94 and a covalent bond; where n is 0, 1 or 2; and R, Rxe2x80x2 and Rxe2x80x3 at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic.
In yet another of its composition aspects, this invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a macrolide antibiotic, aminoglycoside, lincosamide, oxazolidinone, streptogramin, tetracycline or other compound which binds to ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium, and pharmaceutically-acceptable salts thereof.
This invention is also directed to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound of formula I or II.
The multibinding compounds of this invention are effective antibiotics. Accordingly, in one of its method aspects, this invention provides a method for treating bacterial infections.
When used to treat bacterial infections, for example, the method involves administering to a patient having a bacterial infection a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a therapeutically-effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a macrolide antibiotic, aminoglycoside, lincosamide, oxazolidinone, streptogramin, tetracycline or other compound which binds to ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium; and pharmaceutically-acceptable salts thereof.
This invention is also directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties with respect to ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium. The diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarization. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.
This invention is also directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties with respect to bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium. The diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarizability and/or polarization. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.
This invention is also directed to libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties with respect to bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium. These libraries are prepared via the methods described above and permit the rapid and efficient evaluation of what molecular constraints impart multibinding properties to a ligand or a class of ligands targeting the bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium.
Accordingly, in one of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties with respect to bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium which method comprises:
(a) identifying a ligand or a mixture of ligands which bind to bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties.
In another of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties which method comprises:
(a) identifying a library of ligands which bind to bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties.
The preparation of the multimeric ligand compound library is achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands identified in (a) with the linkers identified in (b). Sequential addition is preferred when a mixture of different ligands is employed to ensure heteromeric or multimeric compounds are prepared. Concurrent addition of the ligands occurs when at least a portion of the multimer comounds prepared are homomultimeric compounds.
The assay protocols recited in (d) can be conducted on the multimeric ligand compound library produced in (c) above, or preferably, each member of the library is isolated by preparative liquid chromatography mass spectrometry (LCMS).
In one of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties which library is prepared by the method comprising:
(a) identifying a ligand or a mixture of ligands which bind to bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In another of its composition aspects, this invention is directed to a library of multimeric ligand compounds which bind to bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium which may possess multivalent properties which library is prepared by the method comprising:
(a) identifying a library of ligands which bind to bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In a preferred embodiment, the library of linkers employed in either the methods or the library aspects of this invention is selected from the group comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarizability and/or polarization and amphiphilic linkers. For example, in one embodiment, each of the linkers in the linker library may comprise linkers of different chain length and/or having different complementary reactive groups. Such linker lengths can preferably range from about 2 to 100 xc3x85.
In another preferred embodiment, the ligand or mixture of ligands is selected to have reactive functionality at different sites on the ligands in order to provide for a range of orientations of said ligand on said multimeric ligand compounds. Such reactive functionality includes, by way of example, carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates and precursors thereof. It is understood, of course, that the reactive functionality on the ligand is selected to be complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.
In other embodiments, the multimeric; ligand compound is homomeric (i.e., each of the ligands is the same, although it may be attached at different points) or heteromeric (i.e., at least one of the ligands is different from the other ligands).
In addition to the combinatorial methods described herein, this invention provides for an iterative process for rationally evaluating what molecular constraints impart multibinding properties to a class of multimeric compounds or ligands targeting bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium. Specifically, this method aspect is directed to a method for identifying multimeric ligand compounds possessing multibinding properties with respect to bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium which method comprises:
(a) preparing a first collection or iteration of multimeric compounds which is prepared by contacting at least two stoichiometric equivalents of the ligand or mixture of ligands which target bacterial ribosomal RNA and/or to one or more proteins involved in ribosomal protein synthesis in the bacterium with a linker or mixture of linkers wherein said ligand or mixture of ligands comprises at least one reactive functionality and said linker or mixture of linkers comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand wherein said contacting is conducted under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands;
(b) assaying said first collection or iteration of multimeric compounds to assess which if any of said multimeric compounds possess multibinding properties;
(c) repeating the process of (a) and (b) above until at least one multimeric compound is found to possess multibinding properties;
(d) evaluating what molecular constraints imparted multibinding properties to the multimeric compound or compounds found in the first iteration recited in (a)-(c) above;
(e) creating a second collection or iteration of multimeric compounds which elaborates upon the particular molecular constraints imparting multibinding properties to the multimeric compound or compounds found in said first iteration;
(f) evaluating what molecular constraints imparted enhanced multibinding properties to tile multimeric compound or compounds found in the second collection or iteration recited in (e) above,
(g) optionally repeating steps (e) and (f) to further elaborate upon said molecular constraints.
Preferably, steps (e) and (flare repeated at least two times, more preferably at from 2-50 times, even more preferably from 3 to 50 times, and still more preferably at least 5-50 times.